Cell lines

A549, HT29 and H1299 cell lines

Preparation Method

Cells were treated with 5 μM Thiamet-G for 24 h or the indicated time period.

Reaction Conditions

5μM for 24 hours

Applications

Thiamet G could markedly elevate the O-GlcNAcylation of A549, H1299 and HT29 cells. Thiamet-G treatment did not significantly affect the proliferation of A549, H1299 and HT29. Thiamet-G treatment significantly increased colony formation ability of A549, H1299 and HT29 cells.

Animal models

Six-week-old male Sprague-Dawley rats

Preparation Method

For tau western blot study, rats were treated orally with thiamet-G, by inclusion of inhibitor in the drinking water at a dose of 200 mg kg-1 d-1. Animals were euthanized after one day of treatment. For the thiamet-G dose and time dependency study, six-week-old male Sprague-Dawley rats received single intravenous tail vein injections of either 2, 10 or 50 mg kg-1 and were euthanized at indicated times.

Dosage form

200 mg kg-1 d-1, oralGBP>>2, 10 or 50 mg kg-1 d-1, intravenous injection.

Applications

Thiamet-G is orally bioavailable, thiamet-G can cross the blood brain barrier and inhibit O-GlcNAcase in vivo to cause increased O-GlcNAc levels in brain.

Thiamet G, a potent inhibitor of O-GlcNAcase (Ki =21 nM) was used to increase O-GlcNAcylation levels^[1]. Thiamet G is a stable compound whose fused thiazoline ring system geometrically mimics a transition state of the substrate-assisted enzymatic hydrolysis of protein-O-GlcNAc units and, in this way, effectively inhibits O-GlcNAcase function^[2]. Thiamet-G is orally bioavailable, and thiamet-G can cross the blood brain barrier^[1].

Thiamet G (5 μM, 24h) could markedly elevate the O-GlcNAcylation of human lung epithelial carcinoma A549, non-small cell lung carcinoma H1299 and colon tumor HT29 cells^[3]. Thiamet G (25 μM, 24 h) treated PC-12 cells showed a gradual time-dependent increase in cellular O-GlcNAc levels that reached a maximum after approximately 12 h of exposure^[1].

Thiamet G (2.5 μl of 35 μg/μl) dissolved in 0.9% NaCl was injected bilaterally into the lateral ventricles of the brains at a dose of 175 μg/mouse, RL2 positive bands showed a 5-fold increase in global O-GlcNAcylation 4.5 h after thiamet G injection and a 10-fold increase 24 h after injection^[4]. Thiamet G (500 mg/kg/day p.o.) treatment was able to decrease the number of neurons showing tau pathology, decrease behavioral defects and reduce mice mortality in the Tau.P301L mouse model^[5]. Thiamet-G treated Tau.P301L mice for 3 days in the drinking water (2.5 mg/ml) in the home-cage, improved their breathing deficit in normocapnia and in hypercapnia^[6].

References:
[1]. Yuzwa S A, Macauley M S, Heinonen J E, et al. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo[J]. Nature chemical biology, 2008, 4(8): 483-490.
[2]. Fischer P M. Turning down tau phosphorylation[J]. Nature chemical biology, 2008, 4(8): 448-449.
[3]. Mi W, Gu Y, Han C, et al. O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy[J]. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2011, 1812(4): 514-519.
[4]. Yu Y, Zhang L, Li X, et al. Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation[J]. PloS one, 2012, 7(4): e35277.
[5]. Graham DL, Gray AJ, Joyce JA, Yu D, O'Moore J, Carlson GA, Shearman MS, Dellovade TL, Hering H. Increased O-GlcNAcylation reduces pathological tau without affecting its normal phosphorylation in a mouse model of tauopathy[J]. Neuropharmacology, 2014,1;79:307-13.
[6]. Borghgraef P, Menuet C, Theunis C, Louis JV, Devijver H, Maurin H, Smet-Nocca C, Lippens G, Hilaire G, Gijsen H, Moechars D. Increasing brain protein O-GlcNAc-ylation mitigates breathing defects and mortality of Tau. P301L mice[J]. PloS one, 2013, Dec 23;8(12):e84442.